Publications by authors named "Marca H M Wauben"

66 Publications

Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases.

Vet Pathol 2021 Apr 5:300985821999328. Epub 2021 Apr 5.

8125Utrecht University, Utrecht, the Netherlands.

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.
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http://dx.doi.org/10.1177/0300985821999328DOI Listing
April 2021

Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses.

J Extracell Vesicles 2021 Mar 10;10(5):e12071. Epub 2021 Mar 10.

Department of Biomolecular Health Sciences Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands.

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.
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http://dx.doi.org/10.1002/jev2.12071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944547PMC
March 2021

AFM-Based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles.

Anal Chem 2020 08 13;92(15):10274-10282. Epub 2020 Jul 13.

Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase, Via della Lastruccia 3, 50019 Firenze, Italy.

The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.
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http://dx.doi.org/10.1021/acs.analchem.9b05716DOI Listing
August 2020

Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals.

Cytometry A 2020 06 27;97(6):610-619. Epub 2020 May 27.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.24036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384008PMC
June 2020

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions.

Front Bioeng Biotechnol 2019 12;7:452. Epub 2020 Feb 12.

Consorzio Interuniversitario Nazionale per la Scienza e la Tecnologia dei Materiali, Florence, Italy.

This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs; often referred to as protein contaminants) are present. The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit <50 ng/μl (an order of magnitude higher resolution than that of the Bradford protein assay). For pure solutions, the assay also allows for determining the EV number, as the color shift is linearly dependent on the AuNP/EV molar ratio. Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. The CONAN assay proves to be robust and reliable and displays very interesting performances in terms of cost (inexpensive reagents, run by standard microplate readers), working volumes (1-2 μl of sample required), and time (full procedure takes <1 h). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.
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http://dx.doi.org/10.3389/fbioe.2019.00452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028770PMC
February 2020

The generation and use of recombinant extracellular vesicles as biological reference material.

Nat Commun 2019 07 23;10(1):3288. Epub 2019 Jul 23.

Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, 9000, Belgium.

Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.
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http://dx.doi.org/10.1038/s41467-019-11182-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650486PMC
July 2019

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

J Extracell Vesicles 2018 17;7(1):1473707. Epub 2018 May 17.

Department Laboratory Experimental Clinical Chemistry, Academic Medical Center, University of Amsterdam, DE, Amsterdam, The Netherlands.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.
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http://dx.doi.org/10.1080/20013078.2018.1473707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965025PMC
May 2018

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

PLoS Pathog 2019 02 19;15(2):e1007594. Epub 2019 Feb 19.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50-300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.
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http://dx.doi.org/10.1371/journal.ppat.1007594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396942PMC
February 2019

Extracellular Vesicles in Joint Disease and Therapy.

Front Immunol 2018 12;9:2575. Epub 2018 Nov 12.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.

The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and their linking medium, the synovial fluid, in particular. Further insight is urgently needed in order to appreciate the full potential of EVs and to exploit these in EV-mediated therapies. Physiologic joint homeostasis is a prerequisite for proper functioning of joints and we postulate that EVs play a key role in the regulation of joint homeostasis and hence can have an important function in re-establishing disturbed joint homeostasis, and, in parallel, in the regeneration of articular tissues. In this mini-review EVs in the joint are explained from a historical perspective in both health and disease, including the potential niche for EVs in articular tissue regeneration. Furthermore, the translational potential of equine models for human joint biology is discussed. Finally, the use of MSC-derived EVs that is recently gaining ground is highlighted and recommendations are given for further EV research in this field.
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http://dx.doi.org/10.3389/fimmu.2018.02575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240615PMC
October 2019

Abundantly Present miRNAs in Milk-Derived Extracellular Vesicles Are Conserved Between Mammals.

Front Nutr 2018 18;5:81. Epub 2018 Sep 18.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine Utrecht University, Utrecht, Netherlands.

Mammalian milk is not only a source of nutrition for the newborn, but also contains various components that regulate further development. For instance, milk is an abundant source of microRNAs (miRNAs), which are evolutionary conserved small non-coding RNAs that are involved in post-transcriptional regulation of target mRNA. MiRNAs present in milk can occur in extracellular vesicles (EVs), which are nanosized membrane vesicles released by many cell types as a means of intercellular communication. The membrane of EVs protects enclosed miRNAs from degradation and harbors molecules that allow specific targeting to recipient cells. Although several studies have investigated the miRNA content in milk EVs from individual species, little is known about the evolutionary conserved nature of EV-associated miRNAs among different species. In this study, we profiled the miRNA content of purified EVs from human and porcine milk. These data were compared to published studies on EVs from human, cow, porcine, and panda milk to assess the overlap in the top 20 most abundant miRNAs. Interestingly, several abundant miRNAs were shared between species (e.g., let-7 family members let-7a, let-7b, let-7f, and miR-148a). Moreover, these miRNAs have been implicated in immune-related functions and regulation of cell growth and signal transduction. The conservation of these miRNA among species, not only in their sequence homology, but also in their incorporation in milk EVs of several species, suggests that they are evolutionarily selected to regulate cell function in the newborn.
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http://dx.doi.org/10.3389/fnut.2018.00081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153340PMC
September 2018

Natural T-cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles.

Eur J Immunol 2018 10 17;48(10):1621-1631. Epub 2018 Aug 17.

Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands.

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity.
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http://dx.doi.org/10.1002/eji.201747152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220790PMC
October 2018

Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells.

Cell Mol Life Sci 2018 Oct 28;75(20):3857-3875. Epub 2018 May 28.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.
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http://dx.doi.org/10.1007/s00018-018-2842-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6154026PMC
October 2018

The role of extracellular vesicles when innate meets adaptive.

Semin Immunopathol 2018 09 3;40(5):439-452. Epub 2018 Apr 3.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Innate immune cells are recognized for their rapid and critical contribution to the body's first line of defense against invading pathogens and harmful agents. These actions can be further amplified by specific adaptive immune responses adapted to the activating stimulus. Recently, the awareness has grown that virtually all innate immune cells, i.e., mast cells, neutrophils, macrophages, eosinophils, basophils, and NK cells, are able to communicate with dendritic cells (DCs) and/or T and B cells, and thereby significantly contribute to the orchestration of adaptive immune responses. The means of communication that are thus far primarily associated with this function are cell-cell contacts and the release of a broad range of soluble mediators. Moreover, the possible contribution of innate immune cell-derived extracellular vesicles (EVs) to the modulation of adaptive immunity will be outlined in this review. EVs are submicron particles composed of a lipid bilayer, proteins, and nucleic acids released by cells in a regulated fashion. EVs are involved in intercellular communication between multiple cell types, including those of the immune system. A good understanding of the mechanisms by which innate immune cell-derived EVs influence adaptive immune responses, or vice versa, may reveal novel insights in the regulation of the immune system and can open up new possibilities for EVs (or their components) in controlling immune responses, either as a therapy, target, or as an adjuvant in future immune modulating treatments.
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http://dx.doi.org/10.1007/s00281-018-0681-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208666PMC
September 2018

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Stem Cells Transl Med 2017 08 17;6(8):1730-1739. Epub 2017 Jul 17.

Institute of Medical Biology, A*STAR, Singapore.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.
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http://dx.doi.org/10.1002/sctm.17-0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689784PMC
August 2017

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.

J Extracell Vesicles 2017 7;6(1):1286095. Epub 2017 Mar 7.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University , Utrecht , the Netherlands.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA . These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge - of the nature of EV(-RNA)s and of how to effectively and reliably study them - currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.
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http://dx.doi.org/10.1080/20013078.2017.1286095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345583PMC
March 2017

Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components.

Mol Cell Proteomics 2016 11 6;15(11):3412-3423. Epub 2016 Sep 6.

From the ‡Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands;

Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome.
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http://dx.doi.org/10.1074/mcp.M116.060426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098039PMC
November 2016

Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases.

J Immunol 2016 10 12;197(8):3382-3392. Epub 2016 Sep 12.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, the Netherlands.

Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue-type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9 EV, degranulation was accompanied by the release of CD63 EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63 EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9 EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63 EV from connective tissue-type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.
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http://dx.doi.org/10.4049/jimmunol.1600614DOI Listing
October 2016

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles.

J Extracell Vesicles 2016 9;5:31751. Epub 2016 Aug 9.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands;

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980521PMC
http://dx.doi.org/10.3402/jev.v5.31751DOI Listing
August 2016

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury.

J Cardiovasc Transl Res 2016 08 6;9(4):291-301. Epub 2016 Jul 6.

Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.
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http://dx.doi.org/10.1007/s12265-016-9705-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990609PMC
August 2016

Extending gene ontology in the context of extracellular RNA and vesicle communication.

J Biomed Semantics 2016 12;7:19. Epub 2016 Apr 12.

Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA USA ; Extracellular RNA Communication Consortium (ERCC), ᅟ, ᅟ

Background: To address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research.

Methods: By following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations.

Results: We have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: "extracellular vesicle", "extracellular exosome", "apoptotic body", and "microvesicle" were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO.

Conclusions: As an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search.
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http://dx.doi.org/10.1186/s13326-016-0061-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830068PMC
October 2016

Extracellular vesicles — new tool for joint repair and regeneration.

Nat Rev Rheumatol 2016 04;12(4):243-9

Cell-derived extracellular vesicles (EVs), present in synovial fluid and cartilage extracellular matrix (ECM), are involved in joint development and in the regulation of joint homeostasis. Although the exact function of EVs in these processes remains incompletely defined, the knowledge already acquired in this field suggests a role for these EVs as biomarkers of joint disease, and as a new tool to restore joint homeostasis and enhance articular tissue regeneration. In addition to direct injection of therapeutic EVs into the target site, surface coating of scaffolds and embedding of EVs in hydrogels might also lead to novel therapeutic possibilities. Based on the existing literature of EVs in synovial fluid and articular tissues, and investigation of the molecular factors (including microRNAs) active in joint homeostasis (or during its disturbance), we postulate novel perspectives for the implementation of EVs as a regenerative medicine approach in joint repair.
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http://dx.doi.org/10.1038/nrrheum.2015.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116208PMC
April 2016

Biological properties of extracellular vesicles and their physiological functions.

J Extracell Vesicles 2015 14;4:27066. Epub 2015 May 14.

Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433489PMC
http://dx.doi.org/10.3402/jev.v4.27066DOI Listing
May 2015

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

Cytometry A 2016 Feb 16;89(2):135-47. Epub 2015 Feb 16.

Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible.
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http://dx.doi.org/10.1002/cyto.a.22644DOI Listing
February 2016

Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics.

J Control Release 2015 Feb 30;200:87-96. Epub 2014 Dec 30.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. Electronic address:

Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings. We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.
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http://dx.doi.org/10.1016/j.jconrel.2014.12.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324667PMC
February 2015

Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures.

J Extracell Vesicles 2014 14;3. Epub 2014 Aug 14.

Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at -80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system.
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http://dx.doi.org/10.3402/jev.v3.24215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139932PMC
September 2014

Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells.

Obesity (Silver Spring) 2014 Oct 17;22(10):2216-23. Epub 2014 Jul 17.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands; Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, Utrecht, The Netherlands.

Objective: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined.

Methods: EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry.

Results: In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction.

Conclusions: Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR.
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http://dx.doi.org/10.1002/oby.20847DOI Listing
October 2014

Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages.

Obesity (Silver Spring) 2014 May 9;22(5):1296-308. Epub 2014 Jan 9.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands; Section Metabolic Diseases, Molecular Cancer Research, UMC Utrecht, Utrecht, The Netherlands.

Objective: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined.

Methods: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes.

Results: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR.

Conclusions: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated.
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http://dx.doi.org/10.1002/oby.20679DOI Listing
May 2014

Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

PLoS Biol 2012 18;10(12):e1001450. Epub 2012 Dec 18.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
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http://dx.doi.org/10.1371/journal.pbio.1001450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525526PMC
May 2013

Dynamics of dendritic cell-derived vesicles: high-resolution flow cytometric analysis of extracellular vesicle quantity and quality.

J Leukoc Biol 2013 Mar 17;93(3):395-402. Epub 2012 Dec 17.

Dept. of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Yalelaan 2, Utrecht, The Netherlands.

Nano-sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC-derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC-derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed high-resolution flow cytometry-based method, we can perform a high-throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC-derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS-stimulated DC and CD4(+) T cells affects both the quantity and quality of LPS DC-derived EVs present in the culture supernatant. These data indicate that flow cytometry-based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell-derived EVs.
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http://dx.doi.org/10.1189/jlb.0911480DOI Listing
March 2013